Cytotoxic necrotizing factor type 1 (CNF1) is a member of a family of bacterial toxins that deamidate single glutamine residues in RhoA, Rac, and Cdc42 and thereby constitutively activate these small GTPases. These deamidation events trigger a myriad of effects on the target cells such as actin cytoskeleton rearrangements, cell cycle abnormalities, and alterations in signaling pathways. CNF1 and a membrane-lytic toxin, hemolysin (Hly), are often co- expressed by Escherichia coli strains that cause urinary tract infections (UTIs), i.e., cystitis or pyelonephritis, or acute prostatitis. In fact, the cnf1 locus and a hly operon are co-transcribed and co-regulated from a prototypic uropathogenic E. coli (UPEC) strain during culture in vitro. Of particular relevance to this proposal, CNF1/Hly-producing UPEC isolates are more frequently isolated from humans with blood and high levels of certain pro-inflammatory cytokines in their urine than are toxin-negative UPEC. The latter observation is consistent with our prior findings that CNF1+ UPEC strains elicit a more intense inflammatory response than do isogenic CNF1- mutants in a mouse model of ascending UTI and in a rat model of acute prostatitis and that the CNF1-positive UPEC strain CP9 survives better than does its cnf1 isogenic mutant in human and mouse polymorphonuclear leukocytes (PMNs). We also reported that Hly provokes loss of surface uroepithelial cells in culture and a 3-D organoid model, and we recently found that Hly damages the uroepithelium and evokes hemorrhage in the bladders of mice 24 hours after intraurethral inoculation with CP9. We therefore theorize that CNF1 and Hly enhance the pathogenicity of UPEC strains by: I.) promoting uroepithelial cell shedding and tissue hemorrhage (Hly);ii.) evoking a large influx of potentially tissue-damaging PMNs (CNF1 and Hly) while simultaneously protecting the bacterium from phagocyte-mediated killing (CNF1), and;iii.) eliciting submucosal edema (CNF1).
The specific aims designed to test this hypothesis are to: 1.) delineate the impact of CNF1 and Hly alone and together on the levels of blood, PMNs, and selected pro-inflammatory cytokines in the urine of mice during the first 24 hours after intraurethral infection with CP9 and its cnf1 and hlyA1 single and double mutants and to more broadly compare the host response to these isogenic strains through microarray transcriptional analyses of infected bladders;2.) monitor expression kinetics of cnf1 and the contiguous hly operon by real time RT-PCR in the urine and/or bladders of CP9-challenged mice to ask whether cnf1 and the linked hly operon are co-transcribed in vivo;3.) determine whether CNF1 actually modifies small GTPases in vivo by measuring the extent of activation of Rho, Rac and Cdc42 in uroepithelial cells from bladders of mice infected with CP9 or its CNF mutant;and, 4.) attempt to reduce the extent of inflammation and damage evoked by CP9 through parenteral and/or mucosal immunization of mice with a CNF1/Hly toxoid cocktail.

Public Health Relevance

UTIs are among the most common bacterial diseases of adults, with women affected disproportionately to men. This project will lead to a better understanding of the relative importance of CNF1 and Hly in UPEC-mediated disease and may pave the way for the design of novel preventative and therapeutic strategies against UTIs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI038281-14
Application #
8259452
Study Section
Special Emphasis Panel (ZRG1-HIBP-B (02))
Program Officer
Korpela, Jukka K
Project Start
1995-09-30
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
14
Fiscal Year
2012
Total Cost
$374,888
Indirect Cost
$129,863
Name
Henry M. Jackson Fdn for the Adv Mil/Med
Department
Type
DUNS #
144676566
City
Bethesda
State
MD
Country
United States
Zip Code
20817
Garcia, Tamako A; Ventura, Christy L; Smith, Mark A et al. (2013) Cytotoxic necrotizing factor 1 and hemolysin from uropathogenic Escherichia coli elicit different host responses in the murine bladder. Infect Immun 81:99-109
Grande, Kerian K; Meysick, Karen C; Rasmussen, Susan B et al. (2009) Cytotoxic necrotizing factor type 1-neutralizing monoclonal antibody NG8 recognizes three amino acids in a C-terminal region of the toxin and reduces toxin binding to HEp-2 cells. Infect Immun 77:170-9
Smith, Yarery C; Rasmussen, Susan B; Grande, Kerian K et al. (2008) Hemolysin of uropathogenic Escherichia coli evokes extensive shedding of the uroepithelium and hemorrhage in bladder tissue within the first 24 hours after intraurethral inoculation of mice. Infect Immun 76:2978-90
McNichol, Beth A; Rasmussen, Susan B; Carvalho, Humberto M et al. (2007) Two domains of cytotoxic necrotizing factor type 1 bind the cellular receptor, laminin receptor precursor protein. Infect Immun 75:5095-104
Smith, Yarery C; Grande, Kerian K; Rasmussen, Susan B et al. (2006) Novel three-dimensional organoid model for evaluation of the interaction of uropathogenic Escherichia coli with terminally differentiated human urothelial cells. Infect Immun 74:750-7
McNichol, Beth A; Rasmussen, Susan B; Meysick, Karen C et al. (2006) A single amino acid substitution in the enzymatic domain of cytotoxic necrotizing factor type 1 of Escherichia coli alters the tissue culture phenotype to that of the dermonecrotic toxin of Bordetella spp. Mol Microbiol 60:939-50
Davis, Jon M; Carvalho, Humberto M; Rasmussen, Susan B et al. (2006) Cytotoxic necrotizing factor type 1 delivered by outer membrane vesicles of uropathogenic Escherichia coli attenuates polymorphonuclear leukocyte antimicrobial activity and chemotaxis. Infect Immun 74:4401-8
Davis, Jon M; Rasmussen, Susan B; O'Brien, Alison D (2005) Cytotoxic necrotizing factor type 1 production by uropathogenic Escherichia coli modulates polymorphonuclear leukocyte function. Infect Immun 73:5301-10
Rippere-Lampe, K E; O'Brien, A D; Conran, R et al. (2001) Mutation of the gene encoding cytotoxic necrotizing factor type 1 (cnf(1)) attenuates the virulence of uropathogenic Escherichia coli. Infect Immun 69:3954-64
Meysick, K C; Mills, M; O'Brien, A D (2001) Epitope mapping of monoclonal antibodies capable of neutralizing cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli. Infect Immun 69:2066-74

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